过表达miR-155抑制C2C12成肌分化

Effect of over-expressed miR-155 on inhibiting C2C12 myogenic differentiation

为明确miR-155在C2C12成肌分化中的作用及分子机制,本研究构建了miR-155过表达腺病毒载体,运用过表达miR-155的腺病毒感染C2C12,并诱导其成肌分化。通过形态学观察,成肌标志基因mRNA和蛋白表达水平的检测,以及双荧光素酶报告基因系统对预测的miR-155靶基因(TCF4)的验证,结果表明,C2C12细胞分化中,过表达miR-155明显降低了肌管的形成,成肌标志基因MyoG和MyHC的mRNA表达量极显著地下降(P<0.01),而MyoD差异不显著(P>0.05),成肌标志基因蛋白检测结果与mRNA检测结果一致;进一步研究显示miR-155与预测的TCF4基因的3'UTR 3个靶点(1487-1493,1516-1522,4532-4583)中的1个(4532-4538)结合,并发现过表达miR-155显著降低了TCF4的mRNA水平(P<0.05)。表明miR-155可能通过靶向TCF4抑制C2C12成肌分化。

To clarify the function and molecular mechanism of miR-155 in myogenic differentiation of C2C12, we constructed adenovirus over-expression vector of miR-155, then C2C12 cells were infected by adenovirus and induced myogenic differentiation. First, we observed the morphology of C2C12 after differentiation. Then the mRNA and protein expressions of myogenic markers （MyoD, MyoG and MyHC） were detected by qPCR and western blotting. Subsequently, the dual luciferase reporter gene assay was carried out to validate putative target gene （TCF4） of miR-155. Meanwhile, mRNA level of TCF4 was analyzed after over-expressing miR-155. The results show that over-expressed miR-155 reduced myotubes formation. Moreover, the mRNA and protein expression of MyoG and MyHC decreased significantly （P〈0.01）. Further research demonstrated miR-155 bound the one （4532-4538） of three putative sites （1487-1493,1516-1522,4532-4583） of TCF4 by luciferase reporter gene assay and the mRNA level of TCF4 decreased notably （P〈0.05）. The data suggest that miR-155 inhibited myogenic differentiation of C2C12 through targeted TCF4.